Gardner, RichardGallagher, Pamela Sue2013-07-252015-12-142013-07-252013Gallagher_washington_0250E_11725.pdfhttp://hdl.handle.net/1773/23419Thesis (Ph.D.)--University of Washington, 2013The ubiquitin-proteasome system is one of the principal means for the destruction of misfolded proteins in the cell. In addition to the core enzymes within the ubiquitination cascade and the 26S proteasome, many ancillary factors function to maintain protein homeostasis. For example, Cdc48/p97 is an abundant AAA-ATPase essential for many critical ubiquitin-dependent processes. One well-documented role for Cdc48 is facilitating the delivery of ubiquitinated misfolded endoplasmic-reticulum proteins to the proteasome for degradation. By contrast, Cdc48's participation in misfolded nuclear protein degradation is unknown. In yeast, degradation of misfolded nuclear proteins is mediated by the ubiquitin-protein ligase San1. The results presented here reveal that although Cdc48 and cofactors are involved in the degradation of some San1 substrates, they are not universally required for all San1 substrates. The differential Cdc48 requirement correlates with San1 substrate insolubility: the more insoluble the substrate, the more its degradation requires Cdc48. Expression of Cdc48-dependent insoluble San1 substrates in mutant cdc48 cells results in increased substrate insolubility, greater inclusion formation, and reduced cell viability. We propose that Cdc48 functions to maintain ubiquitinated substrate solubility en route to the proteasome, preventing toxic aggregation from occurring post-ubiquitination. Additionally, the requirement for protein chaperones for San1-mediated degradation also correlates with substrate insolubility. This work illuminates insolubility as a key determinant for Cdc48 and chaperone involvement in the San1 degradation pathway.application/pdfen-USCopyright is held by the individual authors.PharmacologypharmacologyThesis